Therapeutic Malonic Acid/Acetic Acid C60 Tri-Adducts of Buckminsterfullerene and Methods Related Thereto

ABSTRACT

Disclosed and claimed herein are e,e,e malonic acid/acetic acid tri-adduct of buckminsterfullerene of the general formula C 60 R 3 , wherein each R is independently selected from groups of the formula —CR 1 R 2  wherein each R 1  and R 2  is independently selected from the group consisting of —H and —COOH, provided, however, that at least one of the R 1 &#39;s and R 2 &#39;s is a hydrogen. Processes for preparing and uses of the same for treating neuronal injury and for life-extension are also disclosed and claimed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of copending U.S. patentapplication Ser. No. 10/373,425, filed Feb. 24, 2003, now U.S. Pat. No.7,145,032 issued Dec. 5, 2006 which is a continuation-in-part ofapplication Ser. No. 10/083,283; filed Feb. 23, 2002. U.S. patentapplication Ser. Nos. 10/373,425 and 10/083,283 are each incorporatedherein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel C₆₀ derivatives, processes forpreparing such derivatives and methods of treatment. In more detail,disclosed and claimed herein are (a) novel e,e,e malonic acid/aceticacid tri-adducts of buckminsterfullerene and processes for preparing thesame, (b) compositions and methods of treating neuronal injury with atherapeutically effective amount of e,e,e malonic acid/acetic acidtri-adducts of buckminsterfullerene and (c) compositions and methods forprolonging the length or duration of an expected lifespan (referred toalternately as “longevity”) of metazoans or in metazoan cells with atherapeutically effective amount of e,e,e malonic acid/acetic acidtri-adducts of buckminsterfullerene.

2. Related Art

Methods of enhancing the overall health and longevity of humans andtheir companion animals has been a very active area of research. Currentthinking in the field suggests that calorie restriction may help toextend the lifespan of metazoans.

Given the conserved nature of cellular or developmental processes acrossmetazoans, a number of model organisms have been employed to studylongevity, including C. elegans and D. melanogaster.

For example, the genetic analysis of C. elegans has revealed severalgenes involved in lifespan determination. Mutations in Daf-2 (theinsulin receptor) and Clk-1 (“Clock 1”, a gene affecting many aspects ofdevelopmental and behavioral timing) have been shown to extend thelifespan of adults. However, Clk-1 mutants have a higher mortality ratein early life. At later stages of development, the Clk-1 mutants show anincrease in longevity, perhaps by selecting for long-lived individualsin early life. The Clk-1 longevity phenotype is abolished by mutationsin the gene encoding catalase, which is involved in superoxide/freeradical metabolism. Additionally, elimination of coenzyme Q in C.elegans diet has been shown to extend lifespan.

C. elegans harboring mutations in the Eat gene have also shown anincreased longevity, but exhibit decreased food intake and slowedmetabolism. The enhanced longevity associated with this mutation hasbeen attributed to calorie restriction, which has been shown to alsoincrease longevity in metazoans.

In Drosophila, superoxide dismutase (SOD) and catalase over expressionincreased the lifespan of fruit flies by 35%. Mutations also in theMethuselah gene (“Mth”) have been shown to increase lifespan by 20%. Thefunction of Mth, a G-protein coupled receptor, is not known, but mutantshave shown an increased resistance to paraquat (a superoxide radicalinjury inducing agent) toxicity, suggesting it may be a stress-responsegene.

Calorie restriction (CR) has been shown to increase lifespan by 25-35%in all animals studied to date (mice, rats, several species of monkeys,dogs, humans, as well as non-metazoan species such as spiders,Nematodes, and Drosophila). (NB: All animals are metazoans.) However,caloric intake needs to be reduced by as much as 30-40% to achieverobust effects on longevity. Ongoing studies in rhesus and squirrelmonkeys at the National Institute of Aging (“NIA”) (Roth et al., Eur. J.Clin. Nutr. S:157, 2000) found biochemical changes in calorie restrictedmonkeys similar to changes reported in rodents thereby supporting theuniversal nature of calorie restriction on biochemical processes acrossvertebrate species.

Recently, 2-deoxyglucose has been used to produce calorie restrictionwithout limiting oral intake. Animals treated with 2-deoxyglucose havelowered body temperature and decreased plasma insulin levels, similar tochanges observed in calorie-restricted animals (Roth et al., Ann. NYAcad. Sci., 928: 305, 2001). While scientific studies on the effect of2-deoxyglucose on longevity have not been completed, a recent editorialin Science (Feb. 8, 2002) quoted the principal investigator of thesestudies (George Roth, NIA) as saying that one of his monkeys treatedwith 2-deoxyglucose lived 38 months instead of the mean survival of 25months. However, such a claim is not scientifically supported given thesmall sample size. No comment was made on the age of the longest-livedmonkeys in the control populations.

Increases of up to 20% in the expected lifespan of mice has been shownthrough growth factor deprivation, either through genetic manipulationor the administration of growth factor antagonists. Unfortunately,dwarfism is a side effect of growth factor deprivation. In humans,dwarfism, or late-life growth hormone deficiency, appears to reducelongevity, further confusing the issue of whether growth factordeprivation is effective as a means for increasing the duration of theexpected lifespan.

Several papers have indicated that deprenyl (a selective monoamineoxidase (MAO) B inhibitor used to treat Parkinson's disease) increasesthe lifespan of many species. (See, e.g. Knoll, Mech Ageing Dev. 46:237,1988). In one study, chronic treatment of rats with deprenyl from age 96weeks through the end of life “enhanced survival”. Control rats lived147+/−1 weeks, whereas the deprenyl-treated rats lived 198+/−2 weeks.However, the expected mean lifespan for these rats, clearly stated inthe paper, was 182 weeks, so the control group in this study appears tohave had early mortality. Other studies from these laboratories selectedfor high-performing rats, which were then enrolled in the deprenyllongevity studies, thereby potentially artificially skewing the results.

A second study used Fisher 344 rats (Kitani et al., Life Sci 52:281,1993), initiating deprenyl treatment at 18 months of age. The meansurvival of the controls was 28 months, and of the treated animals was30 months, showing an increase in longevity of 7%. However, theseresults were shown to be not statistically significant.

In contrast, another study in Fisher 344 rats with the same dose ofdeprenyl (Carillo et al., Life Sci 67:2539, 2000), observed greatermortality and shortened lifespan in the deprenyl-treated animals.Furthermore, a study from the NIA failed to show any survival benefit inC57B6 mice given chronic deprenyl treatment starting at 18 months of age(Ingram et al., Neurobiol Aging 14:431, 1993). Likewise, a controlledstudy of deprenyl in Drosophila did not show an increase in lifespan(Jordens et al., Neurochem Res 24:227, 1999).

Human trials of deprenyl likewise show conflicting results regardinglongevity. An “open, uncontrolled” trial of deprenyl in Parkinson'spatients showed an increase survival at 9 years (Birkmayer et al., JNeural Transm. 64:113, 1985), although other studies have suggestedincreased mortality in PD patients taking deprenyl, especially inconjunction with L-dopa (e.g. Ben-Shlomo et al., BMJ 316:1191, 1998).

Overall, the data suggest that deprenyl may or may not have weak effectson longevity.

Several genes in mice have been identified as “longevity” genes becausemice with mutations in these genes have greater mean lifespans relativeto the expected lifespan of control mice. These genes include the Amesdwarf mutation, and the Snell dwarf mutation. However, these mutationsresult in small, frail mice which have difficulty feeding. It isbelieved that the longevity conferred by these mutations is essentiallydue to calorie restriction. Recent attempts to use gene array analysis,or other genetic screens for genes associated with longevity phenotypesin worms, flies, and rodents have come up with a number of candidategenes. In general, however, they are frequently “stress-response” genes.

Many compounds, such as Gingko, Ginseng, Vitamin C, have been proposedto improve survival, but controlled and statistically significantsurvival studies reporting the benefit for these compounds are unknown.Vitamin C and a number of drugs reduce the incidence of certain diseaseconditions, e.g., cardiovascular disease, and so, presumably, wouldenhance overall longevity.

Buckminsterfullerene, C₆₀, is a carbon sphere with 12 pentagons and 20hexagons, soluble in aromatic solvents but not in water.

Use of C₆₀(C(COOH)₂)_(n), wherein n is an integer from 1 to 4, isdisclosed for treating neuronal injury in U.S. Pat. No. 6,265,443,issued Jul. 24, 2001 to Choi et al., incorporated herein by reference inits entirety.

The preparation of C₃ hexacarboxylic (“C₃” or “Hexa”) acid reported inthe literature produces mixtures of products, some unidentified, withpoor reproducibility and variable performance on cell culture screening.

SUMMARY OF THE INVENTION

It is in view of the above that the compositions and processes describedand claimed below were developed.

A first embodiment comprises the administration of a composition tometazoans with the result of increasing the metazoan's lifespan, saidcomposition comprising a carboxylated derivative of a C₆₀ fullerene(“carboxyfullerene”), such as a C₆₀ compound having x pairs of adjacentcarbon atoms bonded to a pendant carbon wherein said pendant carbon atomis further bonded to two groups of the general formula —COOH and —R,wherein R is independently selected from the group consisting of —COOHand —H, and wherein x is at least 1.

Another embodiment of a useful compound can be described by the generalformula C₆₀[(CHCOOH)]_(x)[C(COOH)₂]_(y), wherein x is an integer from 0to 3, y is an integer from 1 to 4 and x plus y is an integer from 2 to4.

An additional embodiment is e,e,e malonic acid/acetic acid tri-adductsof buckminsterfullerene of the general formula C₆₀R₃, wherein each R isindependently selected from groups of the formula ═CR¹R² wherein each R¹and R² is independently selected from the group consisting of —H and—COOH, provided, however, that at least one of the R¹'s and R²'s is ahydrogen. More particular embodiments comprise the Penta Pair, TetraQuartet and C₃-lite malonic acid/acetic acid tri-adducts ofbuckminsterfullerene (described in more detail below).

An additional embodiment comprises an e,e,e tri-adduct ofbuckminsterfullerene of the general formula C₆₀R₃, wherein each R isindependently selected from groups of the formula ═CR¹R² wherein each R¹and R² is independently selected from the group consisting of —H, —COOHand —COOMe, provided, however, that at least one of the R¹'s and R²'s isa hydrogen or a —COOMe.

A further embodiment comprises processes of preparing e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene of the generalformula C₆₀R₃, wherein each R is independently selected from groups ofthe formula ═CR¹R² wherein each R¹ and R² is independently selected fromthe group consisting of —H and —COOH, provided, however, that at leastone of the R¹'s and R²'s is a hydrogen, including the Penta Pair, TetraQuartet and C₃-lite e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene.

Yet, additional embodiments comprise the use of novel e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene for treatingneuronal injury and for life-extension (similar to C₃).

It is believed the use of carboxyfullerenes, including e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene, provides asubstantial improvement over calorie restriction as a method whichsubstantially increases the lifespan of metazoans, especially humans,given the inherent difficulties within calorie restriction (including,but not limited to, severe limits to food intake as well as theimpracticability of use with humans in general). It is also shown thatthe novel e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene have similar desirable properties to the C₃ trismalonic acid C₆₀ compound, but have longer half-lives in animals,thereby extending their effective period in vivo. Additionally, becausee,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerene,including Penta-1, Penta-2, the Tetra Quartet and C₃-lite, are morelipophilic than C₃, they can concentrate in lipid-rich tissues, such asbrain.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate various embodiments of the presentinvention and together with the description, serve to explain theprinciples of the invention. In the drawings:

FIG. 1 discloses an analysis of C₃ preparations by HPLC identifyingthree major carboxyfullerene components.

FIG. 2 displays various useful carboxyfullerenes.

FIG. 3 displays the C₃ tris malonic acid regioisomer.

FIG. 4 depicts the survival of C57B6 mice treated with oral C₃ vs.control solution.

FIG. 5 details the configuration of functional groups on e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene.

FIG. 6 depicts structures of C₃-lite (e,e,e tris acetic acid C₆₀) and C₃(e,e,e tris malonic acid C₆₀).

FIG. 7 depicts the structure of Penta-1.

FIG. 8 depicts the structure of Penta-2.

FIG. 9 depicts neuroprotection by Hexa, Penta-1, Penta-2, and C₃-liteversus NMDA toxicity.

FIG. 10 depicts neuroprotection by Hexa, Penta-1, Penta-2, and C₃-liteversus AMPA toxicity.

FIG. 11 details the plasma kinetics of Hexa, Penta-1 and Penta-2, andthe tissue distribution of C₃.

FIG. 12 details the plasma pharmacokinetics and tissue distribution ofC₃-lite.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring to the accompanying drawings in which like reference numbersindicate like elements:

FIG. 1 a discloses an analysis of C₃ preparations by HPLC identifyingthree major components (>99% of the total).

FIGS. 1 b, c indicate all three of the peaks had absorbance spectracharacteristic of e,e,e (C₃) additions to the C₆₀ nucleus indicatingthat the component peaks of C₃ represented e,e,e regioisomers withdifferent headgroups attached to the cyclopropane carbons on C₆₀.

FIGS. 1 c(1)-1 c(3) show compounds 1-3, separated by HPLC, and thendetermined by mass spectrometry to be Hexacarboxylic acid C₃ (1, 80%)and two isomeric Pentacarboxylic acids (2 and 3, 10% each).

FIGS. 1 d(1)-1 d(3) shows mass spectroscopy performed on the Hexa isomer(1), and each Pentacarboxylic acid (2), (3).

FIG. 2 depicts various carboxyfullerenes, including 2 bis isomers, 2tris isomers and a tetra isomer.

FIG. 3 depicts the e,e,e tris malonic acid regioisomer with C₃ symmetry(“C₃”) as both a space filling structure and a chemical structure.

FIG. 4 is a Kaplan-Meier survival curve showing lifespan of C57B6 micetreated with either food coloring (control) or C₃ (0.5 mg/kg/day) intheir drinking water from the age of 12 months through the end of life.The date of spontaneous death for each mouse was recorded, and used tocalculate the lifespan. Lifespan of each mouse was calculated in monthsbecause mice received from the NIA rodent colony have their birth month,but not their specific birth date, recorded. Average survival wascalculated for each treatment and the mean lifespans were compared usinga t-test with significance set to p<0.05 (actual p=0.033). Data from thefirst cohort is graphed by gender (A, B) and combined (C), with eachtreatment group compared to same-sex controls. Weights (g) in treatedand untreated mice (by gender) were not different, e.g., at 19 mos,weights for females: ctrl 27±1, C₃-treated 29±1; for males: ctrl 35±4,C₃-treated 35±6, Mean ±SD.

FIG. 5 details the configuration of functional groups on e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene.

FIG. 6 depicts structures of C₃-lite (e,e,e tris acetic acid C₆₀) andC₃. C₃-lite differs from C₃ in that one of the pair of carboxylic acidsattached to each of the three cyclopropane carbons is replaced with aproton.

FIG. 7 depicts the structure of the Penta-1 compound. HOMO-LUMO energydistribution (top), and ball and stick (bottom). For Penta-1, one of themalonic acid groups of C₃ has been replaced by an acetic acid group. InPenta-1, the proton faces in towards the two malonic acid groups

FIG. 8 depicts the structure of the Penta-2 compound. HOMO-LUMO energydistribution (top), and ball and stick (bottom). For Penta-2, one of themalonic acid groups of C₃ has been replaced by an acetic acid group. InPenta-1, the proton faces away from the two malonic acid groups.

FIG. 9 depicts neuroprotection by the e,e,e malonic acid/acetic acidtri-adducts of buckminsterfullerene Hexa, Penta-1, Penta-2, and C₃-litein cerebrocortical cell cultures versus NMDA receptor-mediatedexcitotoxicity. Cultures were exposed to 200 μM NMDA for 10 minutes,with or without C₃ derivatives (0.5-100 μM). All drugs were washed outafter 10 minutes, and cells were returned to the cell culture incubatorfor 24 hours. Neuronal cell death was then assessed by measuring releaseof lactate dehydrogenase (LDH) by dying neurons. Cell death andprotection was confirmed by imaging propidium iodide staining of deadneurons and by evaluating neuronal morphology using phase contrastmicroscopy. Dose response curves for each compound are shown. Valuesrepresent the % cell death observed in cultures exposed to NMDA alone(with no C₆₀ derivative).

FIG. 10 depicts neuroprotection by the e,e,e malonic acid/acetic acidtri-adducts of buckminsterfullerene Hexa, Penta-1, Penta-2, and C₃-litein cerebrocortical cell cultures versus AMPA receptor-mediatedexcitotoxicity. Cultures were exposed to 6 μM AMPA for 24 hours, with orwithout C₃ derivatives (0.5-100 μM). Neuronal cell death was assessed at24 hours by measuring release of lactate dehydrogenase (LDH) by dyingneurons. Cell death and protection was confirmed by imaging propidiumiodide staining of dead neurons and by evaluating neuronal morphologyusing phase contrast microscopy. Dose response curves for each compoundare shown. Values represent the % cell death observed in culturesexposed to AMPA alone (with no C₆₀ derivative).

FIG. 11 depicts the plasma pharmacokinetics of Hexa, Penta-1, andPenta-2, (A-B) and tissue distribution of Hexa C₃ (C). FIG. 11 (A) is anHPLC analysis of plasma from mice injected with ¹⁴C—C₃ containing 80%Hexa, 10% Penta-1, 10% Penta-2. Samples were t=0 (C₃ sample beforeinjection), t=2 hours (plasma 2 hours after i.p. injection of C₃), t=18hours (plasma 18 hours after injection). FIG. 11 (B) depicts plasmalevels after iv, ip, sc and po administration of C₃. The plasma T_(1/2)for Hexa, Penta-1 and Penta-2 were all 8.2 hours. FIG. 11 (C) depictstissue levels of C₃ at various times after ip administration showingaccumulation through the liver and kidney.

FIG. 12 depicts plasma pharmacokinetics and tissue distribution ofC₃-lite. Figure (A) shows the plasma pharmacokinetics of ¹⁴C₁-C₃-liteand demonstrates a T_(1/2) of 15 h. FIG. 12 (B) depicts the tissuedistribution of C₃-lite after ip administration.

DETAILED DESCRIPTION OF THE INVENTION

The following terminology will be utilized throughout:

“Lifespan” or “expected lifespan,” is the average expected length oflife (from birth to death) that a metazoan would be expected to live(i.e., “generic” expected lifespan) in a particular environment if thatmetazoan were not treated with carboxyfullerenes.

“Malonic acid/acetic acid C₆₀ derivatives” are acetic acid derivativesof C₆₀, malonic acid derivatives of C₆₀ and mixed malonic acid/aceticacid derivatives of C₆₀.

“Hexa” and C₃ represent C₃(C₆₀(C(COOH)₂)₃, where the malonic acid groupsare all at the e,e,e positions (compound 1, below).

“Pentas” or “Penta Pair” means (C₆₀(C(COOH)₂)₂(C(CHCOOH)), where theR-groups are attached to cyclopropane carbons at the e,e,e positions.There are two stereoisomers Penta-1 and Penta-2 (compound 2, below).

“Tetras” or “Tetra Quartet” means (C₆₀(C(COOH)₂)(C(CHCOOH))₂, whereinacetic acid/malonic acid groups are attached to cyclopropane carbons atthe e,e,e positions. There are four stereoisomers (compound 3, below).

“C₃-lite” means (C₆₀(CHCOOH))₃ wherein the acetic acid groups areattached to cyclopropane carbons at in the e,e,e positions. There arefour stereoisomers (compound 4, below).

“An e,e,e malonic acid/acetic acid tri-adduct of buckminsterfullerene”or “malonic acid/acetic acid C₆₀ derivative” means abuckminsterfullerene with three pendant groups independently selectedfrom malonic groups (>C(COOH)₂) and acetic groups (>CHCOOH) at the e,e,epositions; to with:

Compound R-Groups 1 R₁ = R₂ = R₃ = R₄ = R₅ = R₆ = COOH (C₃) 2 R₁ = H, R₂= R₃ = R₁ = R₅ = R₆ = COOH (Penta Pair) 3 R₁ = R₃ = H, R₂ = R₄ = R₅ = R₆= COOH (Tetra Quartet) 4 R₁ = R₃ = R₅ = H, R₂ = R₄ = R₆ = COOH (C₃-lite)5 R₁ = R₂ = COOt-Bu, R₃ = R₄ = R₅ = R₆ = COOMe 6 R₁ = R₂ = R₃ = R₄ =COOt-Bu, R₅ = R₆ = COOMe 7 R₁ = R₂ = COOH, R₃ = R₄ = R₅ = R₆ = COOMe 8R₁ = R₂ = R₃ = R₄ = COOH, R₅ = R₆ = COOMe 9 R₁ = H, R₂ = COOH, R₃ = R₄ =R₅ = R₆ = COOMe 10 R₁ = R₃ = H, R₂ = R₄ = COOH, R₅ = R₆ = COOMeCarboxyfullerenes, Including e,e,e Malonic Acid/Acetic Acid Tri-Adductsof Buckminsterfullerene, for Prolonging the Expected Length or Durationof a Lifespan

Many important biological reactions generate reactive oxygen speciesintentionally, or as unwanted toxic by-products. While reactive oxygenspecies, including superoxide (O₂ ^({dot over (−)})) and hydrogenperoxide (H₂O₂), are harnessed for specific physiological functions,they also pose an ongoing threat to the viability and integrity of cellsand tissues. In response, cells and organisms have developed a varietyof mechanisms to defend themselves against O₂ ^({dot over (−)}) andH₂O₂. In metazoans, O₂ ^({dot over (−)}) is removed by twometallo-enzymes, Cu, Zn-superoxide dismutase (SOD1), and MnSOD (SOD2).H₂O₂, in turn, is removed by catalase, a heme iron containingmetallo-enzyme, or glutathione peroxidase, a family of proteins whichutilize selenocysteines in conjunction with glutathione to convert H₂O₂to O₂ and H₂O. However, these endogenous antioxidant defense systems maybe overwhelmed under pathological conditions. This has led to attemptsto develop additional antioxidants (useful substances that inhibitoxidation or inhibit reactions promoted by oxygen or peroxides) as smallmolecules to supplement the antioxidant defenses of cells as potentialtherapeutic agents.

A number of water-soluble C₆₀ derivatives (superoxidedismutase-mimetics) retain the antioxidant properties of their parentfullerene molecule, allowing its free radical scavenging abilities to beexploited in biological systems and thereby act as agents which reducecell damage and death.

One group of C₆₀ derivatives, carboxyfullerenes, act as a decompositioncatalyst for H₂O₂ and O₂ ^({dot over (−)}). Although,manganese-containing protoporphyrin compounds, including MnTMPyp, havebeen reported to act as decomposition catalysts for O₂^({dot over (−)})/H₂O₂, these compounds rely on oxidation-reduction ofthe manganese atom to catalyze decomposition. It has been now discoveredby the inventors that although C₃ is a non-metallic compound, it toopossesses similar catalytic properties. It is believed this compound isthe first non-metallic compound to act in such a manner.

Because many important biological reactions generate reactive oxygenspecies intentionally, or as unwanted toxic by-products, antioxidantmolecules capable of supplementing the antioxidant defenses of cells aspotential therapeutic agents are therapeutically useful. This includescarboxyfullerenes such as e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene. These novel carboxyfullerene compositions haveantioxidant properties. Through our research, the reactivity of C₃ withO₂ ^({dot over (−)}) and H₂O₂ was characterized. The K_(i) of C₃ for O₂^({dot over (−)}) was calculated to be 3×10⁶ M⁻¹sec⁻¹. Analysis of C₃after interaction with O₂ ^({dot over (−)}) and H₂O₂ indicated that nopermanent chemical or structural changes occurred at either the C₆₀moiety or the malonic acid groups, supporting the claim that C₃ is atrue catalyst. Although, manganese-containing protoporphyrin and salencompounds have also been reported to act as catalysts for thedecomposition of O₂ ^({dot over (−)})/H₂O₂, these compounds rely onoxidation-reduction of the metal atom to catalyze decomposition, whereasthe malonic acid fullerene derivatives do not require a metal atom tocatalyze the decomposition of reactive oxygen species.

Thus, in view of the foregoing discovery, carboxyfullerenes, includinge,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerene areuseful in the elimination of reactive oxygen species, especiallyreactive oxygen species that are physiologically relevant, such ashydrogen peroxide (H₂O₂) and superoxide (O₂ ^({dot over (−)})).

Described and claimed herein are methods of increasing a metazoan'sexpected lifespan by administering therapeutically effective amounts ofantioxidants which result in an extended metazoan, or metazoan's cell,lifespan. In particular, compositions comprising the antioxidantcarboxyfullerenes are used as treatments to increase the lifespan ofmetazoans or metazoan cells.

The compounds useful herein are thus carboxyfullerene compounds, theircorresponding salts and esters having x pairs of adjacent carbon atomsof the C₆₀ fullerene bonded to at least one pendant carbon, wherein thependant carbon atom is further bonded to two groups of the generalformula —COOH and —R, wherein R is independently selected from the groupconsisting of —COOH and —H, and wherein x is at least 1. Examples ofisomers of this general formula are shown in FIGS. 1-3. Also usefulherein are e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene of the general formula C₆₀R₃, wherein each R isindependently selected from groups of the formula ═CR¹R² wherein each R¹and R² is independently selected from the group consisting of —H and—COOH, provided, however, that at least one of the R¹'s and R²'s is ahydrogen.

Thus, provided is a method of extending the expected lifespan ofmetazoans or metazoan cells, including mammals and more particularly,humans, by administering to the metazoan an e,e,e malonic acid/aceticacid tri-adduct of buckminsterfullerene of the general formula C₆₀R₃,wherein each R is independently selected from groups of the formula═CR¹R² wherein each R¹ and R² is independently selected from the groupconsisting of —H and —COOH, provided, however, that at least one of theR¹'s and R²'s is a hydrogen.

All carboxyfullerene compounds, including the e,e,e malonic acid/aceticacid tri-adducts of buckminsterfullerene, can be administeredsystematically as a composition containing the active compound and apharmaceutically acceptable carrier compatible with said compound. Inpreparing such a composition, any conventional pharmaceuticallyacceptable carrier may be utilized. When the drug is administeredorally, it is generally administered at regular intervals.

In therapeutic use, e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene may be administered by any route whereby drugs areconventionally administered. Such routes include intravenous,intramuscular, subcutaneous, intrathecal, intraperitoneal, topical, andoral.

Pharmaceutical compositions comprising the e,e,e malonic acid/aceticacid tri-adducts of buckminsterfullerene of this invention can be madeup in any conventional form, including a solid form for oraladministration such as tablets, capsules, pills, powders, granules, andthe like. These pharmaceutical compositions may be sterilized and/or maycontain adjuvants such as preservatives, stabilizers, wetting agents,emulsifiers, salts for varying the osmotic pressure, and/or buffers.

Typical preparations for intravenous administration would be sterileaqueous solutions including water/buffered solutions. Intravenousvehicles include fluid, nutrient and electrolyte replenishers.Preservatives and other additives may also be present such asantibiotics and antioxidants.

The e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerenedescribed and claimed herein are useful in pharmaceutically acceptableoral modes. These pharmaceutical compositions contain said compound inassociation with a compatible pharmaceutically acceptable carriermaterial. Any conventional carrier material can be utilized. Anyconventional oral dosage form such as tablets, capsules, pills, powders,granules, and the like may be used. The carrier material can be anorganic or inorganic inert carrier material suitable for oraladministration. Suitable carriers include water, gelatin, gum arabic,lactose, starch, magnesium stearate, talc, vegetable oils,polyalkylene-glycols, petroleum jelly and the like. Furthermore, thepharmaceutical composition may contain other pharmaceutically activeagents. Additional additives such as flavoring agents, preservatives,stabilizers, emulsifying agents, buffers and the like may be added inaccordance with accepted practices of pharmaceutical compounding.

An oral dosage form may comprise, for example, tablets, capsules of hardor soft gelatin, methylcellulose or of another suitable material easilydissolved in the digestive tract. The oral dosages contemplated willvary in accordance with the needs of the individual patient asdetermined by the prescribing physician. An example of this oral dosageform embodiment comprises capsules or tablets containing from 50 to 500mg of e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene.

Methods of treatment with e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene of this invention can generally be given to adultsdaily, preferably orally, intramuscularly, subcutaneously orintravenously. If intramuscularly, intravenously or subcutaneously,treatments should be given in an amount from as low as about 0.1 mg/kgto an amount as high as 3 mg/kg, with the precise dosage being varieddepending upon the needs of the patient. The daily dose, if givenorally, would be expected to be as little as 0.1 mg/kg to an amount ashigh as 15 mg/kg. In general, this therapy may be carried outprophylactically for an indefinite time.

The e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullereneof the present invention may be administered chronically (e.g., daily)or frequently (e.g., once a week). The C₃ Hexa isomer, the Penta Pair,the Tetra Quartet and C₃-lite are expected to be the more effectiveagents. The expected daily dose of the C₃ isomer, if given byintravenous, intramuscular or subcutaneous delivery, would be about 0.1mg/kg to about 3 mg/kg. The daily does if given orally would be expectedto range between 0.1 mg/kg and 15 mg/kg.

The above dosing information is based on a pharmokinetics study carriedout in mice, toxicity testing in mice and toxicity testing in rats. Inmice, the plasma half-lives of C₃, Penta-1 and Penta-2 were calculatedto be about 8 hours, while the plasma half-life of C₃-lite was about 15hours. The 50% lethal dose (LD50) for a single injection of thecarboxyfullerenes was >70 mg/kg. C₃, Penta-1 and Penta-2 were clearedfrom mice by excretion through both the liver and kidney while C₃-litewas cleared through the liver and fat. Using calculations based on thispharmacokinetic data, the therapeutic plasma levels appear to be between0.1 and 1 μg/ml. Although equivalent amounts of carboxyfullerenes areabsorbed if the compound is given by intravenous, intraperitoneal orsubcutaneous administrations, only about 1/15^(th) of this dose isabsorbed when given orally (e.g., in drinking water). However, thestandard pharmaceutical formulations of carboxyfullerenes for oraldelivery are expected to significantly increase the bioavailability oforally-administered carboxyfullerenes (e.g., incorporation ofcarboxyfullerenes into time-release tablets). Additionally, e,e,emalonic acid/acetic acid tri-adducts of buckminsterfullerene are morelipophilic than C₃, which allows them to concentrate in lipid-richtissues, such as brain.

It is envisioned that these claimed processes are useful for allmetazoans, including but not limited to vertebrates, and morespecifically to mammals, including humans and their companion animals.

The lifespan increased by carboxyfullerenes is the expected averagelength of time (from birth to death) that a metazoan would be expectedto live, if that metazoan were not treated with carboxyfullerenes. Asthe results of Example 2 and FIG. 4 indicate, mice subject to thistreatment had an actual lifespan of 28.7 months, which corresponded to alifespan that is about 20% greater than the control mouse's lifespan of23.5 months. The lifespan of the control mouse used in this examplerepresents the generic “expected lifespan.”

Nonlimiting embodiments of compounds useful for extending the durationof life of a mammal, comprise compounds such as e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene of the generalformula C₆₀R₃, wherein each R is independently selected from groups ofthe formula ═CR¹R² wherein each R¹ and R² is independently selected fromthe group consisting of —H and —COOH, provided, however, that at leastone of the R¹'s and R²'s is a hydrogen. Nonlimiting examples of e,e,emalonic acid/acetic acid tri-adducts of buckminsterfullerene includecompounds selected from the group consisting of the Penta Pair, theTetra Quartet, C₃-lite, their stereoisomers, mixtures thereof and thelike.

A further nonlimiting embodiment is a process for extending the lifespanof a metazoan or metazoan cells comprising administering to saidmetazoan a composition comprising at least one e,e,e malonic acid/aceticacid tri-adduct of buckminsterfullerene of the general formula C₆₀R₃,wherein each R is independently selected from groups of the formula═CR¹R² wherein each R¹ and R² is independently selected from the groupconsisting of —H and —COOH, provided, however, that at least one of theR¹'s and R²'s is a hydrogen. Nonlimiting examples of e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene include compoundsselected from the group consisting of the Penta Pair, the Tetra Quartet,C₃-lite, their stereoisomers, mixtures thereof and the like. It isenvisioned that said composition would comprise at least one e,e,emalonic acid/acetic acid tri-adduct of buckminsterfullerene, itspharmaceutically acceptable salts and pharmaceutically accepted esters,and a pharmaceutically acceptable carrier, wherein the components are insaid composition in a therapeutically effective amount. The e,e,emalonic acid/acetic acid tri-adduct of buckminsterfullerene can beadministered intravenously, intramuscularly, subcutaneously or orally.This process can be used with all metazoans, including but not limitedto vertebrates, mammals and humans.

Malonic Acid/Acetic Acid Tri-Adducts of Buckminsterfullerene

C₆₀ e,e,e malonic acid/acetic acid tri-adducts display additionaldesirable qualities including increased water solubility. Several novelC₆₀ e,e,e malonic acid/acetic acid tri-adducts have been synthesized andcharacterized, including: 1) acetic acid derivatives of C₆₀, and 2)e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerene. Twocompounds (2), referred to as “Penta-1” (FIG. 7) and “Penta-2” (FIG. 8)are e,e,e derivatives of C₆₀ with two malonic acid groups, and oneacetic acid group added to C₆₀. Penta-1 and Penta-2 differ in whetherthe proton attached to the cyclopropane carbon of the acetic acid groupfaces in toward the two malonic acid groups, or away from them. A secondset of four compounds “the Tetra-Quartet” (3) has one malonic acidgroup, and two acetic acid groups in the e,e,e positions. A third set ofcompounds are the e,e,e tris acetic acid derivative of C₆₀ (C₃-lite) (4)(FIG. 6). These e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene include Penta-1, Penta-2, the four Tetra's and theC₃-lite compounds, their pharmaceutically acceptable salts and esters.

A general formula for e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene comprises C₆₀R₃, wherein each R is independentlyselected from groups of the formula ═CR¹R² wherein each R¹ and R² isindependently selected from the group consisting of —H and —COOH,provided, however, that at least one of the R⁵'s and R²'s is a hydrogen.

Neuroprotective e,e,e Malonic Acid/Acetic Acid Tri-Adducts ofBuckminsterfullerene

Because many important biological reactions generate reactive oxygenspecies intentionally, or as unwanted toxic by-products, antioxidantmolecules capable of supplementing the antioxidant defenses of cells aspotential therapeutic agents are therapeutically useful.

The e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerenederivatives described and claimed herein may be used to prevent, treator ameliorate the progression of any disease condition caused by freeradicals, especially when the free radicals are released as a result ofglutamate neurotoxicity (“excitotoxicity”). Treating excitotoxic injurymeans reducing the extent of damage to central neurons which have beendamaged by glutamate released from surrounding cells. Neurotoxic eventssuch as excitotoxicity can occur during many types of acute neurologicalinsults such as hypoxia/ischemia, such as occurs during stroke,hypoglycemia, epilepsy or trauma. Neurotoxic events may also be involvedin chronic neuronal damage caused by neurodegenerative disorders such asHuntington's disease, Alzheimer's disease, amyotropic lateral sclerosis(“ALS”), and the neurodegenerative effects of AIDS. Thus, e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene also are useful inmethods of treating diseases in which a neurotoxic injury occurs.

Arachidonic acid (“AA”) is released in neurons due to an influx ofexcessive Ca²⁺ into the neuronal cells which is caused by NMDA receptorstimulation by glutamate (the glutamate having been released by neuronswhich were damaged by the neurotoxic event, itself). The excessive Ca²⁺influx activates phospholipase A₂, a calcium-dependent enzyme whichbreaks down cell membranes liberating the AA. The metabolism of AA byendogenous lipoxygenases and cyclooxygenases leads to the production ofthe oxygen free radicals that trigger peroxidative degradation ofneuronal lipid membranes which results in the neuronal damage or death.Therefore, reducing oxygen-derived free radicals by administering acomposition comprising a free radical scavenging e,e,e malonicacid/acetic acid tri-adduct of buckminsterfullerene provides analternative mechanism by which glutamate-induced neurotoxicity isinhibited.

Similar to the above processes and compositions for increasing thelifespan of individuals, compositions and methods for treatingneurotoxic injury in a patient suffering a neurotoxic injury comprisesadministering to said patient a composition comprising a therapeuticallyeffective amount of e,e,e malonic acid/acetic acid tri-adducts ofbuckminsterfullerene to that individual. More in particular, theseembodiments comprise the administration of at least one e,e,e malonicacid/acetic acid tri-adduct of buckminsterfullerene of the generalformula C₆₀R₃, wherein each R is independently selected from groups ofthe formula ═CR¹R² wherein each R¹ and R² is independently selected fromthe group consisting of —H and —COOH, provided, however, that at leastone of the R¹'s and R²'s is a hydrogen. A further embodiment comprisesthe administration of C₃-lite, Penta-1, Penta-2 and the Tetra Quartet tothe patient. Methods of treatment with these novel compounds can beintravenous, intramuscular, subcutaneous or through oral delivery.

A first embodiment comprises compounds for the treatment of neuronalinjury. Nonlimiting examples of such compounds include e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene of the generalformula C₆₀R₃, wherein each R is independently selected from groups ofthe formula ═CR¹R² wherein each R¹ and R² is independently selected fromthe group consisting of —H and —COOH, provided, however, that at leastone of the R¹'s and R²'s is a hydrogen. Additional nonlimiting examplesof e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullereneinclude compounds selected from the group consisting of the Penta Pair,the Tetra Quartet, C₃-lite, their stereoisomers, mixtures thereof andthe like.

A second embodiment comprises a method of treating neurotoxic injury ina patient suffering a neurotoxic injury by administering to said patienta composition comprising an e,e,e malonic acid/acetic acid tri-adduct ofbuckminsterfullerene of the general formula C₆₀R₃, wherein each R isindependently selected from groups of the formula ═CR¹R² wherein each R¹and R² is independently selected from the group consisting of —H and—COOH, provided, however, that at least one of the R¹'s and R²'s is ahydrogen, its pharmaceutically acceptable salts and pharmaceuticallyacceptable esters, and a pharmaceutically acceptable carrier, whereinsaid compound is present in said composition in an amount effective totreat said neurotoxic injury. Nonlimiting examples of e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene useful for thetreatment of neurotoxic injury include e,e,e malonic acid/acetic acidtri-adducts of buckminsterfullerene selected from the group consistingof the Penta Pair, the Tetra Quartet, C₃-lite, their stereoisomers,mixtures thereof and the like.

As indicated above, the compounds of this method can be administered inamounts from about 1.5 mg/kg to about 1500 mg/kg daily or from about 10mg/kg to about 60 mg/kg daily.

A third embodiment is a method of inhibiting neurotoxic injury in apatient where said injury is caused by free radical oxygen speciesreleased by neurons due to the stimulation by glutamate of NMDAreceptors of said neurons by administering to said patient a compositioncomprising an e,e,e malonic acid/acetic acid tri-adduct ofbuckminsterfullerene of the general formula C₆₀R₃, wherein each R isindependently selected from groups of the formula ═CR¹R² wherein each R¹and R² is independently selected from the group consisting of —H and—COOH, provided, however, that at least one of the R¹'s and R²'s is ahydrogen, its pharmaceutically acceptable salts and pharmaceuticallyacceptable esters, and a pharmaceutically acceptable carrier, whereinsaid compound is present in said composition in an amount effective toinhibit said neurotoxic injury. Nonlimiting examples of e,e,e malonicacid/acetic acid tri-adducts of buckminsterfullerene useful for thisprocess include compounds selected from the group consisting of thePenta Pair, the Tetra Quartet, C₃-lite, their stereoisomers, mixturesthereof and the like.

The e,e,e malonic acid/acetic acid tri-adducts of buckminsterfullerenecan be administered in amounts from about 1.5 mg/kg to about 1500 mg/kgdaily, or in an amount from about 10 mg/kg to about 60 mg/kg daily.

Chemical Synthesis of e,e,e Malonic Acid/Acetic Acid Tri-Adducts ofBuckminsterfullerene

The preparation of C₃ reported in the literature produces mixtures ofproducts, some unidentified, with poor reproducibility and variableperformance on cell culture screening. Therefore, more reliable anddirect processes to specific components are necessary.

Methods of Synthesizing High Concentrations of Hexa

Using the below methods of synthesis, large quantities of Hexa can begenerated, with results generally greater than 90, 94 and 97%.

One such process involves preparing an initial solution of water,methanol and C₃ methyl ester in a solvent. The mole ratio of water toester is between about 100:1 to about 20:1. Solvents useful fordissolving the C₃ methyl ester include aromatic solvents such astoluene, t-butyl acetate in toluene or the like. The initial solution isthen mixed thoroughly (approximately 1 hour) and may appear cloudy atfirst. Sodium hydroxide, up to about 1M, in methanol is next added,after which the solution is stirred vigorously until no color remains insolution (up to about 2 hours). To ensure the reaction is complete, TLCcould be used.

Once the reaction is complete, water is added to form a nonaqueous layerand an aqueous layer. The layers are then separated by any standardmethod including but not limited to decanting, using a separatoryfunnel, or any other equipment useful for separating layers. Anyresidual solvent is then stripped in vacuo. The aqueous layer, whichcontains the product, is heated in an inert atmosphere (N₂ or the like)at about 60° C. for about 1 to about 2 hours.

The isomer distribution can be determined utilizing HPLC protocols 1 and2 (below) and generally is above 90, 94 or 97%.

Direct Synthesis of the Penta Pair or Tetra Quartet By Decarboxylation

The Penta Pair or the Tetra Quartet are directly synthesized byinitially adding an ester of bromomalonate (including, but not limitedto dimethyl bromomalonate) to di-t-butylmalonyl C₆₀ (as prepared byBingel, U.S. Pat. No. 5,739,376, incorporated herein by reference in itsentirety) in an aromatic solvent chosen to maximize solubility, such astoluene or benzene, followed by a tertiary amine, including but notlimited to 1,8-diazabicyclo (5,4,0) undec-7-ene (“DBU”) or sodiumhydride (prepared as disclosed in Bingel '376 at Col. 4, lines 44-45) toform a reaction mixture. To prepare the Penta Pair, these reactants arein a 1:2:2 C₆₀ to malonate to base ratio concentration. To prepare theTetra Quartet, a 1:1:1 ratio is utilized. A suitable time (about 30minutes) should be allowed to pass to ensure thorough mixing of thereaction mixture. The end point of the reaction can be determined byTLC. The reaction mixture is next poured onto a column of silica gel inthe solvent selected above. To separate the bis isomers, the columnshould be eluted with the solvent until all of the bis isomers come off.The solvent should then be changed to ethyl acetate/solvent mixtures andshould be added in increasing concentrations of ethyl acetate, such as0.5% to 1% to about 2%. These mixtures will elute the following fourester components: D₃ (trans-3, trans-3, trans-3 tris malonic acid C₆₀);e,trans-3,trans-2; e,trans-4,trans-3; and C₃ esters. The eluted C₃ esterfractions are then evaporated in vacuo.

An acid miscible in the above solvent, including but not limited top-toluenesulfonic acid monohydrate, trifluoroacetic acid (TFA) ormethane sulfuric acid, is next added to a solution of the C₃ esterfractions in an aromatic solvent (including but not limited to toluene)to dissolve the fractions. This solution is heated to about 88-89° C.Lower temperatures can be used, but the reaction will proceed at aslower rate. If TFA is used, room temperature should be sufficient. Oncethe reaction has begun (after about 45 minutes, or determined viaassay), additional acid should be added. A precipitate will beginforming after about 10 minutes. Heating continues until the reaction iscomplete (about 90 minutes or determined by assay such as TLC or HPLC).The solvent is next removed from the precipitate. Water and ethylacetate are added to the solids to form an ethyl acetate/water mixture.The ethyl acetate solution is separated and then washed with water toremove the catalyst acid. If necessary, the ethyl acetate should bewashed multiple times. The ethyl acetate is then evaporated in vacuo toleave a solid.

The solid is then dissolved in about a 1:1:2 acetonitrile:water:acetonemixture. Other ratios can be utilized to maximize solubilizing thematerial, including using acetone alone. The mixture is heated via anoil bath, heating mantle or the like to about 50° C., about 76.5° orabout 100° C. to complete the reaction. To ensure the reaction iscomplete, an assay could be run. A quantity of acetone is next added tosufficiently dissolve the precipitate. Heating is continued for about 5hours to about 7.5 hours. Any volatile solvents are then removed (forexample, in vacuo) and ethyl acetate is then added to form a homogeneoussolution. This solution is then evaporated in vacuo to produce a solid.

This solid is next dissolved in aromatic solvents (such as toluene or 5%t-butyl acetate in toluene (1 mg/ml)) containing methanol and water.Various ratios can be used (such as a 100:4.4:0.2); however, a limitedquantity of water should be used to avoid producing two phases. Thissolution is then mixed well, with stirring for about an hour. Aftermixing, a 20 to 1 ratio of 1M sodium hydroxide in methanol is added.After complete precipitation (once the color completely drops out whichcould take about one hour to about two hours), water is added. Thesolvent layers are then thoroughly separated, ensuring any residualsolvent is removed. The aqueous layer is heated at about 60° C. to about110° C. for about two hours plus or minus half an hour. An assay couldbe run to determine that the reaction is completed.

After chilling, a strong acid, such as sulfuric acid, (one that will notbe extracted into ethyl acetate) is added to the solution in a quantitysufficient to neutralize the base from above. The final product isextracted with ethyl acetate as described above.

Direct Synthesis of C₃-Lite (in Solution or Neat).

C₃-lite can be directly synthesized by heating either dry or dissolvedsamples of C₃ derivatives of Hexa, Penta-1 and Penta-2. If dissolvedsamples are used, the derivatives should be dissolved in mixtures ofacetonitrile:water. Any standard method of heating can be used,including but not limited to a heating mantel, oil bath or the like. Theresulting solution/sample is heated for a sufficient time to produceC₃-lite (less than about 24 hours) at about 60° C. to about 70° C. toabout 81° C. (the boiling point of acetonitrile). The solvents are thenremoved in vacuo to give a solid product.

When a neat sample of C₃ derivatives is utilized, the sample is heatedat about 150° C. in a vacuum oven at about −30 mm Hg.

Thermal Decomposition of C₃ in Solution to Generate Pentas, Tetras, orC₃-Lite.

A product comprising the Pentas, Tetras, or C₃-lite is produced bydissolving a sample of C₃ derivatives, containing Hexa, Penta-1 andPenta-2 in a 1:1 ratio of acetonitrile:water. The resulting solution isheated to about 60° C. After about 1.5 hours of heating, the Pentaconcentration is near its maximum. The longer heat is applied (about 3.5to about 5.5 hours), the Penta concentration will decrease while theconcentration of C₃-lite and Tetras will increase.

When the desired results are achieved, the solvents are removed in vacuoto give a solid product.

Utilizing NaOMe/MeOH to synthesize a mixture of Hexa and Penta.

Sodium methoxide or sodium hydroxide is added to a solution ofe,e,e-tris dimethylmalonyl fullerene (“C₃ ester”) in an aromatic solventunder an inert gas such as N₂, to form an initial solution. The aromaticsolvent can be selected from the group consisting of, but not limitedto, toluene and t-butyl acetate in toluene. The sodium methoxide is in a16-20:1 ratio with the C₃ ester. A red-orange precipitate will beginforming immediately. After a suitable time, about 1 to 2 hours, water isadded to form an aqueous and nonaqueous layer. Any color will move intothe aqueous layer. The layers are then separated. The aqueous layer ischilled to 0-5° C., in an ice bath or the like, then acidified withexcess sulfuric acid to a pH of about 2. This acidic solution is thenextracted with ethyl acetate to transfer all of the color to the ethylacetate extracts. The combined ethyl acetate extracts are next washedwith water to remove any yellow contaminants. This solution is thenevaporated and dried in vacuo at about room temperature.

Solutions for cell screening are prepared by dissolving the solid in 0.1N sodium hydroxide. Sufficient base can be added to neutralize allcarboxyls. The actual concentration can be determined by assay such asuv using 4400 for the extinction coefficient, determined on the esterprecursor in toluene. HPLC protocol 1 can be used to determine percentof Hexa, Penta-1 and Penta-2. The three components can then be separatedby HPLC.

The data herein demonstrate that the disclosed carboxyfullerenes are anovel class of antioxidants with the unique ability to decomposeoxygen-derived free radicals, and that these compounds have unusualbroad and powerful capabilities to extend the lifespan of individuals.

Further features and advantages of the above compositions and processes,as well as the structure and operation of various embodiments, aredescribed in detail below with reference to the accompanying drawings.

The above disclosure describes several preferred embodiments which arenot scope limiting in any manner. The skilled artisan in the practice ofthese processes and compositions will recognize other embodiments thatare not overtly disclosed herein. The embodiments above are furtherillustrated by the examples described below. These examples are meant toillustrate these embodiments and are not to be interpreted as scopelimiting in any manner.

All of the references and related art cited herein represent a portionof the present state of the art and are therefore incorporated herein intheir entirety.

EXAMPLE 1 Preparation of C₃ carboxyfullerenes

Materials. Silica gel (Merck grade 9385, 260-400, 60 A) was obtainedfrom Aldrich Chemicals (St. Louis, Mo.). Other reagents were purchasedfrom Sigma Chemical Co. (St. Louis, Mo.) and other standard sources.

The C₃ regioisomer of malonic acid C₆₀ (e,e,e C₆₀[C(COOH)₂]₃) wassynthesized by dissolving C₆₀ (720 mg, 1.00 mmol) in toluene at aconcentration of 1 mg/ml by stirring overnight. Dimethyl bromomalonate(632.4 mg, 2.69 mmol) was added, followed by 1,8-diazabicyclo (5.4.0)undec-7-ene (DBU, 493 mg, 3.24 mmol). The reaction mixture was stirredfor 2 hours, filtered through a pad of silica gel and concentrated invacuo. The residue was chromatographed on a 450 ml column of silica gel(Merck, 280-400 mesh), starting in toluene. The colored components wereseparated by adding increasing amounts of ethyl acetate (EtOAc) to thetoluene. The C₃ fraction eluted in 5% EtOAc in toluene. Purity of theC₆₀ malonic ester fractions was monitored by TLC and HPLC. The C₃ ester(0.25 g, 0.23 mmol) was dissolved in toluene (250 ml) and sparged withnitrogen. Addition of sodium methoxide (2.22 ml of 2.2 M, 4.88 mmol)resulted in a precipitate within minutes. The mixture was stirred atroom temperature under nitrogen for one hour. Water (20 ml) was addedand the mixture was stirred overnight. All colored products went intothe water layer. The layers were separated, and the aqueous layer waschilled and acidified with 20% sulfuric acid (1.32 ml). The solution wasextracted with EtOAc three times, resulting in all color going into theorganic layer. The organic layer was washed several times with water toextract a yellow contaminant. The EtOAc layer was then evaporated, andthe residue 223.2 mg (89% of theoretical) was freeze-dried.

EXAMPLE 2 Experimental Method for Longevity Trial with Mice

Twelve month old C57B6NIH male and female mice (equal numbers) werepurchased from the National Institute on Aging (NIA) Aging RodentColony. Mice shipped from this colony were not selected in any way forhealth, tumors or other disabilities, and all mice obtained from thecolony were subsequently enrolled in the study. Mice were randomlyplaced in same-sex numbered cages, two per cage, ear-punched foridentification, and weighed. Mice were then trained on a rotorod twiceper week for three sessions, and were then tested on the rotorod inthree sessions to measure motor performance at baseline. Cages were thenassigned to receive either treatment A or treatment B by an observer whowas blind to what these treatments would be.

Treatment A was a solution of C₃ (28.75 μM) in water, and treatment Bwas commercial food coloring added to match the red C₃ solution.Solution A or solution B was placed in the water bottles, and solutionswere topped-off twice weekly, and filtered to remove any particulatesbiweekly by an individual blind to the identity of the solutions. At 19months of age, mice were weighed again, and underwent another round ofrotorod training and testing. Mice were allowed to die spontaneously,and their date of death were recorded by the Animal Housing Facilitystaff as part of the normal operating procedure of the facility.Facility staff believed that animals were on an antibiotic solution, anddid not know the purpose of the study. When animals died, the cagenumber, identity of the animal, and the date of death were recorded onthe death notice, which was then sent to the laboratory, where theinformation was entered into the database.

The results of these experiments are displayed in FIG. 4 and show amarked increase (approximately 20%) in the lifespan of mice. Inaddition, because longevity was increased by the oral dosing of a drug,it is the first practical method for achieving increased longevity inmetazoans. The increased lifespan of C₃-treated mice was not accompaniedby a reduction in weight.

EXAMPLE 3 Toxicity Study Utilizing Rats

Rat toxicity testing of C₃ was also carried out with two strains of rats(Sprague-Dawley and Long-Evans) which received up to 10 mg/kg day for 30days without showing any toxicity (i.e., decreased survival, impairedgrooming or decreased feeding).

EXAMPLES 4-13

Identified below are novel several e,e,e malonic acid/acetic acidtri-adducts of buckminsterfullerene. Major components from preparing C₃Hexa include two isomeric pentacarboxylic acids in approximately equalabundance (the Penta Pair), minor products including four isomeric tetraacids (the Tetra Quartet) and four isomeric tri-decarboxylation products(C₃-lite). These products are less soluble in water than C₃ (morelipophilic) which may increase these novel compounds absorption andretention in tissues.

The processes below gives a concentration of 94+% hexacarboxylic acidand provides directed routes to the other novel components. Mixturesrich in the Penta Pair, Tetra Quartet and C₃-lite were obtained bydecarboxylation. The Penta Pair and Tetra Quartet were also obtained viaan alternative strategy using t-butyl protection.

HPLC Methods for Analysis of Derivatives

All of the following HPLC methods used a Hewlett Packard/Agilent 1100series HPLC with a quaternary pump and diode array detector. Separationswere performed on either Zorbax SB-C18 4.6×250 mm column (5 μm packing)(column A) or Zorbax SB-C8 4.6×250 mm column (5 μm packing) (column B),maintained at room temperature. All methods used a solvent flow rate of1 ml/min.

Protocol 1—Analysis of Hexa and Penta Compounds.

Solvents were 0.1% TFA in water (solvent A) and 0.1% TFA in 95%acetonitrile and 5% water (solvent B). Samples were eluted from thecolumn using a gradient from 40:60; A:B, to 10:90; A:B, over 15 min.,with an additional 15 minutes at 10:90; A:B. Compounds were monitoredand identified by their UV-vis absorbance using an in-line diode arraydetector.

Protocol 2—Alternative Method to Separate Hexa and Penta Compounds.

The HPLC solvents were 0.1% TFA in water (solvent A) and 0.1% TFA in 95%2-propanol and 5% water (solvent B). Compounds were eluted from (columnA or B) using a gradient from 95:5; A:B, to 52:48; A:B over 10 minutes,the gradient progressed further to 51:49; A:B over 10 minutes then to21:79; A:B over 5 minutes. After that it remained isocratic for anadditional 2 minutes.

Protocol 3—Method to Separate the Tetra Quartet.

Preparations of the Tetra Quartet were eluted from (column A or B)using: 0.1% TFA in water (solvent A) and 0.1% TFA in 95% acetonitrileand 5% water (solvent B). The solvent composition was maintained at30:70; A:B, for 35 minutes, followed by a gradient to 5:95; A:B over 2min., followed by an additional 10 minutes at 5:95; A:B.

Protocol 4—Analysis of C₃-Lite.

Preparations of C₃-lite were analyzed using solvents as described forprotocol 1 with either (column A or B). The gradient was modified tostart at 95:5; A:B, maintained for 5 minutes, followed by gradientchange to 5:95; A:B over 30 min. The solvent composition remained at5:95; A:B for an additional 35 min.

Protocol 5—Analysis of t-butyl, Methyl Esters.

The solvent was 50% acetonitrile and 50% dichloromethane (solvent A).Samples were eluted from (column A) with an isocratic program of 100%(solvent A) over 5 minutes.

Protocol 6—Analysis of partial methyl esters.

The HPLC solvent was 0.1% TFA in 95% acetonitrile and 5% water (solventA). Samples were eluted from (column A or B) in an isocratic 100%(solvent A) solution over 120 min.

EXAMPLE 4 NaOMe/MeOH Method to Synthesize a Mixture of Hexa and PentaIsomers

Sodium methoxide (1.84 mL of 2.2M, 4.05 mmol) was added to a solution ofe,e,e-tris dimethylmalonyl fullerene (C₃ ester) (224.1 mg, 0.202 mmol)in 224 mL of toluene under N₂. Precipitation of a red-orange solid beganimmediately. Water was added after one hour and all of the color wentinto the aqueous layer. The layers were separated and the aqueous layerwas chilled, acidified with sulfuric acid (1.10 mL of 3.7 M, 4.07 mmol)and extracted with ethyl acetate (2×40 ml and 1×10 mL). The combinedethyl acetate extracts were washed with 3×40 mL of water which removedthe yellow contaminants. Evaporation and drying in vacuo at roomtemperature afforded 199.2 mg (96.1% of theory based on Hexa carboxylicacid). Solutions for cell screening were prepared by dissolving thesolid in 0.1 N sodium hydroxide to give a solution of approximately 25mM by weight. The actual concentration was then determined by uv using4400 for the extinction coefficient, determined on the ester precursorin toluene. The UV-vis λ max (nm) was 488, the max/min ratio (488/414nm) was 2.19. Using HPLC protocol 1, it was determined that thesynthesis produced 65.1% Hexa isomer, 14.3% Penta-1 and 20.6% Penta-2.The three components were separated by HPLC, and identified by massspectrometry (Table 3) and ¹H NMR (Table 4).

EXAMPLE 5 Toluene 6% MeOH, and Equal Amounts of Water and NaOH toProduce 95% Hexa

A solution of water (0.216 mL, 12 mmol), methanol (17.1 mL) and C₃methyl ester (667 mg, 0.601 mmol) in 500 mL of toluene was stirred for 1h. The solution appears cloudy at first. If base is added immediatelythe yield of Hexa is lower. Sodium hydroxide in methanol (12.9 mL of0.93 M, 12.0 mmol) was added. After 1.5 h, there was no ester remainingin solution, as assessed by TLC and the color of the solution. Water,100 mL, was added. The toluene layer was separated and the aqueous layerwas concentrated to remove methanol and toluene, then heated for 2 h at60° under N₂ to complete the hydrolysis. Workup by extraction gave 490.8mg (79.5% theoretical yield). The UV-vis λ max was 487 nm, and themax/min ratio (487/411 nm) was 3.10. Utilizing HPLC protocol 1, it wasdetermined that the isomer distribution was 95.2% Hexa, 1.7% Penta-1 and3.0% Penta-2.

EXAMPLE 6 Method Using Equal Amounts NAOH and Water, and 2% MEOH MixedTogether Before Addition, with 5% T-Butyl Acetate in Toluene to Generate88% Hexa, and the Penta Isomers

Sodium hydroxide (3.679 g, 0.092 mol) was dissolved in 92 mL methanoland 1.655 mL (0.092 mol) of water to give a 0.982 M solution. A portionof this solution (12.3 mL, 12.0 mmol) was added to C₃ methyl ester (670mg, 0.604 mmol) dissolved in 585 mL of toluene containing 5% t-butylacetate (1.14 mg/mL compared to 1.0 mg/mL) at room temperature under N₂.A precipitate slowly formed. After 1 h, water was added to the reactionmixture. The phases were separated and the aqueous layer wasconcentrated to remove methanol and toluene, then heated at 60° for 2 h.The product, 573.3 mg (92.5% theoretical), was recovered from theaqueous layer by ethyl acetate extraction as described above. The UV-visλ max was 489 nm, and the max/min ratio (489/411 nm) was 3.37. UtilizingHPLC protocol 1, it was determined that the isomer distribution was88.4% Hexa, 5.6% Penta-1 and 6.0% Penta-2.

EXAMPLE 7 Method Using t-butyl acetate w/5 Water Molecules to Generate96% Hexa

A solution containing water (1.52 mL, 84.4 mmol), methanol (40 mL) andC₃ methyl ester (0.845 mmol by UV) in 950 mL of 5% t-butyl acetate wasstirred for 30 min under N₂. Sodium hydroxide in methanol (16.9 mL of 1N, 16.9 mmol) was added. After 2 h, all color was in the precipitate.Water, 50 mL, was added and the toluene was decanted and residualtoluene was stripped in vacuo. The aqueous layer was heated for 2 h at600 under N₂. Workup by extraction gave 798.1 mg (92.1% theoretical).The UV-vis λ max was 489 nm, and the max/min ration (489/412 nm) was2.71. Utilizing HPLC protocol 1, it was determined that the isomerdistribution was 96.7% Hexa and 1.5% of each Penta.

The total methanol concentration is 6%. If base is added immediately, orif base and all of the methanol are combined before addition to theester, the isomer distribution shifts to lower Hexa content (see Example6).

EXAMPLE 8 Alternative Synthesis of 97% Hexa Using Toluene Only

This procedure was the same as Example 7 except toluene was used insteadof 5% t-butyl acetate in toluene. Utilizing HPLC protocol 1, it wasdetermined that the product had an isomer distribution of 97.2% Hexa,1.3% Penta-1 and 1.5% Penta-2. The UV-vis max was 491 nm, and themax/min ratio (491/412 nm) was 2.97, and the yield was 90.5%. ¹³C NMR(K₂CO₃, D₂O, 600 MHz): δ 173.84, 173.76, 152.55, 151.33, 149.49, 149.41,149.05, 148.98, 148.92, 148.78, 148.61, 148.13, 147.96, 146.55, 146.46,145.89, 144.81, 143.82, 143.07, 142.99, 80.69, 79.88, 69.55.

EXAMPLE 9 Penta Pair Direct Synthesis

Step 1—Dimethyl bromomalonate (281.3 mg, 1.20 mmol) was added todi-t-butylmalonyl C₆₀ (0.592 mmol) in 200 mL of toluene followed by DBU(202.4 mg, 1.33 mmol) (Bingel '376 at Col. 4, lines 44-45). After 30min, the reaction mixture was poured onto a 410 mL (4×310 cm) column ofsilica gel in toluene. The column was eluted with toluene until all ofthe bis isomers (25% by UV-vis) came off. The solvent was changed toethyl acetate/toluene mixtures (0.5% to 2%). Ester preparations enrichedin the following components eluted (using HPLC protocol 5): D₃ (trans-3,trans-3, trans-3 tris dimethyl malonyl C₆₀); e,trans-3,trans-2 trisdimethyl malonyl C₆₀; e,trans-4,trans-3 tris dimethyl malonyl C₆₀; andC₃. The C₃ ester fractions (HPLC protocol 1) were evaporated in vacuo.Mass spec. and ¹H NMR data are in Tables 3 and 4.

Step 2—p-Toluenesulfonic acid monohydrate (22.5 mg, 0.118 mmol) wasadded to a solution of the C₃ fractions in toluene (15 mL) and placed inan oil bath at 88-89°. After 45 min, additional p-toluenesulfonic acidmonohydrate (15.6 mg, 0.082 mmol) was added. Product began precipitatingwithin 10 min. Heating was continued for 90 min. The toluene was removedfrom the precipitate and water and ethyl acetate were added to thesolids. The ethyl acetate was washed three times with water to removep-toluenesulfonic acid. The ethyl acetate was evaporated in vacuoleaving 118.2 mg of solid. Compounds were analyzed using HPLC Protocol6.

Step 3—The solid was dissolved in 10 mL of 1:1:2acetonitrile:water:acetone. A sample, 0.2 mL, was removed for analyses.The remainder was heated in an oil bath at 76.50. After 30 minprecipitate began to separate. Acetone (10 mL) was added and the solidsdissolved. Heating was continued and the decarboxylation was 73%complete after 1.75 h. After heating for a total of 7.5 h, the volatilesolvents were removed in vacuo and ethyl acetate was added to give ahomogeneous solution. A portion was removed for analyses (1.9 mg). Theremainder was evaporated in vacuo to give 0.13 g of solid. Compoundswere analyzed by HPLC protocol 1.

Step 4—The above solid (0.125 mmol) was dissolved in toluene (120 mL)containing methanol (4.9 mL) and water (0.128 mL, 7.1 mmol) and stirredfor one h before addition of sodium hydroxide in methanol (2.3 mL of 1.0M, 2.3 mmol). Solid began precipitating within 5 min. Water, 50 ml, wasadded after one h. The toluene was separated and the aqueous layerstripped of methanol and toluene and heated at 60° for two h. Sulfuricacid (0.621 mL of 3.7 M, 2.3 mmol) was added to the chilled solution andthe product was extracted using ethyl acetate as described above. Thedry weight was 91.1 mg. U-vis λ max(nm). Compounds were analyzed usingHPLC protocol 1. Mass spec. and ¹H NMR data are in Tables 3 and 4. ¹³CNMR (K₂CO₃, D₂O, 600 MHz): δ 175.39, 175.36, 173.94, 173.88, 173.82,173.80, 173.74, 173.68, 173.65, 153.39, 152.46, 152.32, 152.23, 152.15,151.37, 151.22, 149.66, 149.46, 149.38, 149.24, 149.14, 149.04, 148.97,148.86, 148.55, 148.45, 148.42, 148.36, 148.35, 148.13, 147.96, 147.78,147.04, 146.96, 146.87, 146.70, 146.66, 146.60, 146.42, 146.38, 146.03,145.94, 145.70, 145.55, 145.22, 144.86, 144.79, 144.67, 144.58, 144.00,143.92, 143.88, 143.81, 143.76, 143.62, 143.07, 143.02, 142.98, 142.93,142.11, 142.05, 80.72, 80.69, 80.58, 79.97, 79.75, 79.67, 77.21, 77.12,76.38, 76.30, 69.52, 69.44, 69.38, 69.72.

EXAMPLE 10 Synthesis of the Tetra Quartet

Step 1—Dimethyl bromomalonate (38 mg, 0.16 mmol) was added to a sampleof bis-di-t-butyl malonates of C₆₀ (0.167 mmol by UV) in 30 mL oftoluene. DBU (28 mg, 0.18 mmol) was added and the reaction was followedby TLC (2% EtAc in toluene on silica gel). The reaction was completeafter one h. The reaction mixture was poured onto a 330×2.5 cm column ofsilica gel in toluene. Bis fractions (12%, four components, HPLCprotocol 5) were recovered in toluene and then the eluant was changed to0.5% ethyl acetate in toluene. Fractions, including esters of D₃,e,trans-3,trans-2, e,trans-4,trans-3 and C₃ were obtained. The C₃fraction was 88.5% pure by HPLC protocol 1, and was used without furtherpurification. UV-vis (0.5% EtAc in toluene) λmax was 486 nm, the max/minratio was 3.57.

Step 2—P-Toluenesulfonic acid hydrate (4.9 mg, 0.0258 mmol) was added toC₃ ester (0.0154 mmol) in 4.5 mL of toluene and the mixture was placedin an oil bath at 77°. The temperature was raised to 85° over 15 min andthen more p-toluenesulfonic acid (5.9 mg, 0.0310 mmol) was added. Within5 min, precipitation began. Heating was continued for 30 min. Themixture was cooled and the toluene was decanted. Addition of water andethyl acetate produced a two phase mixture with all of the color in theethyl acetate layer. The ethyl acetate was washed three times with waterand evaporated to give 18.3 mg of solid. Compounds were also analyzedusing HPLC protocol 1.

Step 3—The solid above was dissolved in 5 mL of 4:1 acetonitrile:waterand 0.546 mL was removed for analyses. The remainder was heated in anoil bath at 74°. After 65 min, solids were present. Addition of 3 mL ofacetone gave a homogeneous solution and heating was continued for 5.5 h.A portion, 0.8 mL, of the solution, was removed for analyses and theremainder, 7.0 mL, was concentrated to remove acetone and acetonitrile.HPLC (95:5:0.1,ACN:H₂O:TFA, min,%) 19.341 (26.2), 38.180 (25.1, 43.798(22.2), and 80.874 (21.6). Compounds were also analyzed using HPLCprotocol 1.

Step 4—Sodium hydroxide, (0.59 mL Of 0.1 n, 0.059 mmol) was added to thesuspension obtained above in about 3 mL of water. The solids dissolved.Sodium hydroxide (0.6 mL of 1N, 0.6 mmol) was added and the solution washeated at 62° for one h. HPLC indicated complete hydrolysis. Thesolution was chilled and acidified with sulfuric acid (0.162 mL of 3.7M). Extraction with ethyl acetate afforded 11.4 mg of solid. HPLC(95:5:0.1, ACN:H₂O:TFA, min,%) 6.076 (26.4), 10.784 (24.5), 13.883(26.0), 23.589 (23.1). Compounds were analyzed using HPLC protocol 1.Mass spec. and ¹H NMR data are in Tables 3 and 4.

EXAMPLE 11 Direct Synthesis of C₃-Lite in Solution

A sample of C₃ (329.7 mg, 0.32 mmol, 71% Hexa, 15% Penta 1 and 14% Penta2) was dissolved in 6 mL of 1:1 acetonitrile:water and placed in an oilbath at 70° C. The progress of the reaction was monitored by HPLC(Protocol 2). After 2.25 h, the reaction was nearly complete. Solidsbegan precipitating after 5 h. Heating was continued for 24 h. Solventswere removed in vacuo to give 199.3 mg of product which had thefollowing composition by HPLC protocol 1: C₃-lite peak 1 (14.0%), peak 2(39.4%), peak 3 (37.0%), peak 4 (8.7%). LC-MS pos m/e was 895 for each,neg m/e was 1007 for each (M+TFA-H). All had the UV-vis spectrum of C₃(e,e,e additions to C₆₀). ¹H NMR and mass spec data are in Tables 3 and4. The solid was soluble in sodium hydroxide, moderately soluble inacetone and ethyl acetate, poorly soluble in water, methanol andacetonitrile.

EXAMPLE 12 Thermal Decomposition of C₃ in Solution to Generate Pentas,Tetras, or C₃-Lite

A sample of C₃ containing 97.4% Hexa, 1.2% Penta 1 and 1.3% Penta 2(74.8 mg) was dissolved in 10 mL of 1:1 acetonitrile:water and heated at60° C. The composition was monitored by HPLC, using protocols 2 and 4(see Table 1).

TABLE 1 HPLC analysis of products produced by thermal decomposition ofC₃ in solution. Retention Times Incubation Time, h Identification (min)0 1.5 3.5 5.5 Hexa 10.8 **97.4% 40.5 9.2 2.6 Penta 1 13.0 1.2 23.1 20.713.7 Tetra 15.6 — 3.5 9.5 12.2 Tris 18.9 — — 1.3 2.6 Penta 2 19.3 1.323.7 24.0 17.9 Tetra 22.1 — 3.1 9.0 12.0 Tetra 26.2 — 3.4 10.6 14.0Tetra 32.0 — 2.8 2.9 6.6 Tris 39.0 — — 9.2 13.4 Tris 56.9 — — 2.3 4.4Tris 73.9* — — — — Sums Hexa 40.5 9.2 2.6 Pentas 46.8 44.7 31.6 Tetras12.8 38.3 51.6 Tris — 6.5 13.6 *not obtained. **All values are percentof injected compound.

EXAMPLE 13 Neat Decarboxylation of C₁ to C₃-Lite

A sample of C₃ containing 93.8% Hexa, 3.5% Penta 1 and 2.7% Penta 2 wasplaced as a dry powder in a vacuum oven at 150° C. and −30 mm of Hg. Thecomposition was monitored by HPLC protocol 2. Table 2 shows thetime-dependent production of decarboxylation products of C₃, includingC₃-lite (See Table 2).

TABLE 2 HPLC analysis of products produced by neat thermal decompositionof C₃. Retention Time Incubation Time, h Identification (min) 0 19 64168 Hexa 28.9 **93.8% — — — Penta-1 30.2 3.5 — — — Tetra-1 31.9 — — — —Penta-2 33.2 2.7 — — — Tris-1 34.1 — 13.2 13.1 13.0 Tetra-2 34.4 — — — —Tetra-3 35.5 — — — — Tris-2 37.0 — 36.5 36.6 36.6 Tetra-4 37.5 — — — —Tris-3 39.9 — 37.0 37.0 37.1 Tris-4 43.4 — 13.4 13.2 13.3 Sums Hexa 93.8— — — Penta pair 6.2 — — — Tetra Quartet — — — — C₃-lite — 100 100 100**All values are percent of injected compound.

TABLE 3 Mass Spectral Data. Compound numbers refer to those listedabove. M/e, M/e Base Compound Mass spec expected found peak Fragment 1QTOF ES− 1025 1025 1025 2 ″ 981 981 981 M-2CO₂/2 3 ″ 937 937 893 —CO₂ 4″ 893 893 893 5 FAB+ 1195 1195 720 C(CO₂Me)₂ 6 ″ 1279 1279 720 7 QTOFES− 1081 1081 1038 —CO₂ 8 ″ 1053 1053 1009 —CO₂ 9 ″ 1037 1037 1037 10 ″965 965 965

TABLE 4 ¹H NMR Data¹ Compound Solvent δCH² δCH δMethyl δt-Butyl 2DMSO-d₆ 4.894(0.53) 4.750}0.46) — — 4.734 3 ″ 4.887(0.47) 4.739(0.53) —— 4.870 4.721 4 ″ 4.881(0.56) 4.730(0.44) — — 4.863 4.726 4.861 4.7114.707 5 CDCl₃ — — 3.875(4) 1.505(3) 3.832(2) 1.487(6) 3.824(2) 1.475(3)6 ″ — — 3.872(1) 1.503(3) 3.829(1) 1.491(3) 1.487(3) 1.473(3) 7Acetone-d₆ 3.949(1) 3.889(1) 8 ″ 3.948(1) 3.887(1) 9 DMSO-d₆ 4.838(1)4.676(1) 3.902(6) 3.843(6) 10 ″ 4.806(1) 4.658(1) 3.896(3) 3.8853.836(3) 3.829 ¹Varian Unity 300 NMR Spectrometer ²Chemicalshift(integration)

TABLE 5 Carbon-Hydrogen analysis Combustion Analysis Calculated FoundCompound C H C H Hexa C₃ (run 1) C₆₉H₆O₁₂ × 3H₂O 76.68 1.12 76.88 1.7673.55 1.73 Hexa C₃ (run 2) C₆₉H₆O₁₂ × 4H₂O* 75.42 1.28 75.15 1.96 74.991.94 C₃ lite C₆₆H₆0₆ × 2.5H2O 84.35 1.18 84.78 1.18 *7.41% wt loss ondrying

TABLE 6 Chemical properties of C₃ and C₃-lite Properties ThermalSolubil- Stability² ity³ Com- Acidity¹ t_(1/2) min, # of Water, SodiumHydration pound [H⁺/A⁻] 60° products mg/ml salt⁵ of Na salt⁴ C₃ 3.3 6010 >454⁶ >450⁶ 1.31 C₃ lite — — — insoluble   9 4.36 ¹Measured pH and uvin 1:1 acetonitrile:water. All solutions were 1.29-1.97 × 10 − 4mmol/mL. ²In 1:1 acetonitrile:water. ³The concentration was calculatedfrom the uv spectrum measured on the liquid portion of a saturatedsolution. ⁴Sodium salts were freeze dried at room temperature for 4days. This value in the dry weight divided by the expected weight forcomplete neutralization. ⁵Calculated for the acid. ⁶Solution too dark tosee undissolved solids.

TABLE 7 Visible spectroscopic properties ¹ Ester ² Acid ³ Solution HPLCSolution HPLC εmax εmax εmax εmax Literature values⁴ Compound λ nm εεmin λ nm εmin λ nm εmin λ nm εmin λ nm ε C₃ max 485 4400 4.55 482 4.85487 3.7 484 5.08 481 ⁵ 4000 min 409 967 408 409 408 486 ⁶ 3270 ¹Solution spectra measured on Beckman DU-600 in 1 cm cells. HPLC valuesfrom Agilent diode array detector in acetonitrile: water: 0.1 TFAgradients. ² Measured on methyl esters in toluene ³ Measured on halfneutralized acids in water ⁴ Dichloromethane, no data given for minima ⁵U. Reuther, T. Brandmuller, W. Donaubauer, F. Hampel, A. Hirsch, Chem.Eur. J. 2002, 2261-2273 ⁶ G. Rapenne, J. Crassous, L. E. Echegoyen, L.Echegoyen, E. Flapan, F. Diederich, Helv. Chim. Acta. 2000, 83,1209-1223

In view of the foregoing, it will be seen that the several advantages ofthe above embodiments and examples are achieved and attained. Theembodiments were chosen and described in order to best explain theprinciples of the invention and its practical application to therebyenable others skilled in the art to best utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated.

As various modifications could be made in the constructions and methodsherein described and illustrated without departing from the scope of theinvention, it is intended that all matter contained in the foregoingdescription or shown in the accompanying drawings shall be interpretedas illustrative rather than limiting. For example, the process asdescribed above could easily be applied to other metazoans, includingbut not limited to humans, with the same results. Thus, the breadth andscope of the present invention should not be limited by any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims appended hereto and theirequivalents.

1.-21. (canceled)
 22. A process for extending the lifespan of amammalian cell comprising administering to a mammal a compositioncomprising an e,e,e malonic acid/acetic acid tri-adduct ofbuckminsterfullerene of the formula C₆₀(R)₃ represented by the followingstructure:

wherein each R is independently selected from the group consisting ofmoieties of the formula ═CR¹R², wherein each R¹ and R² of said ═CR¹R²moiety is independently selected from the group consisting of —H and—COOH, provided, however, that at least one R¹ or R² of at least one ofsaid ═CR¹R² moieties is a hydrogen, and wherein the C in each of said═CR¹R² moieties is bonded directly to two adjacent carbons of the C₆₀moiety.
 23. The process of claim 22 wherein said e,e,e malonicacid/acetic acid tri-adduct of buckminsterfullerene is selected from thegroup consisting of C₆₀(C(COOH)₂)₂(CHCOOH), C₆₀(C(COOH)₂)(CHCOOH)₂, andC⁶⁰(CHCOOH₃.
 24. The process of claim 22 wherein said compositioncomprises said e,e,e malonic acid/acetic acid tri-adduct ofbuckminsterfullerene, its pharmaceutically acceptable salts andpharmaceutically accepted esters, and a pharmaceutically acceptablecarrier, present in said composition in a therapeutically effectiveamount.
 25. The process of claim 24 wherein said e,e,e malonicacid/acetic acid tri-adduct of buckminsterfullerene is administeredintravenously, intramuscularly, subcutaneously or orally.
 26. Theprocess of claim 25 wherein said e,e,e malonic acid/acetic acidtri-adduct of buckminsterfullerene is administered intravenously,intramuscularly or subcutaneously in an amount of at least 0.1 mg/kg.27. The process of claim 26 wherein said e,e,e malonic acid/acetic acidtri-adduct of buckminsterfullerene is administered intravenously,intramuscularly or subcutaneously in an amount of about 3 mg/kg.
 28. Theprocess of claim 25 wherein said e,e,e malonic acid/acetic acidtri-adduct of buckminsterfullerene is administered orally in an amountof at least 0.1 mg/kg.
 29. The process of claim 25 wherein said e,e,emalonic acid/acetic acid tri-adduct of buckminsterfullerene isadministered orally in an amount of about 15 mg/kg.
 30. The process ofclaim 25 wherein said e,e,e malonic acid/acetic acid tri-adduct ofbuckminsterfullerene is administered daily.
 31. (canceled)
 32. Theprocess of claim 1 wherein said mammal is a human.
 33. The process ofclaim 1 wherein said mammal is a companion animal. 34.-69. (canceled)